Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in water and soluble in alcohol. Commercially viable quantities of isoprene can be obtained by direct isolation from petroleum C5 cracking fractions or by dehydration of C5 isoalkanes or isoalkenes (Weissermel and Arpe, Industrial Organic Chemistry, 4th ed., Wiley-VCH, pp. 117-122, 2003). The C5 skeleton can also be synthesized from smaller subunits. It would be desirable, however, to have a commercially viable method of producing isoprene that was independent of nonrenewable resources.
Isoprene monomer is employed in the manufacture of polyisoprene and various copolymers (with isobutylene, butadiene, styrene, or other monomers). Building a strain (prokaryotic or eukaryotic) capable of producing commercially viable levels of isoprene requires optimization of part of or the entire DXP or MVA pathway or both MVA and DXP pathways. A key enzyme in the pathway is isoprene synthase (IspS), which converts the precursor DMAPP to isoprene. Isoprene synthases (IspS) that have been identified include those from plants such as poplar, English oak and kudzu vine. Some of the plant IspS enzymes identified have been partially characterized in part by expression in E. coli and some of the kinetic parameters of these enzymes have been determined in vitro with purified protein. However, the solubility of the native IspS enzymes and even some of the recombinant enzymes are insufficient for commercial production of isoprene in a biological host. Thus, one problem to be solved is the provision of isoprene synthase variants (e.g. with substitutions at specific residues) which have improved solubility such that a greater amount of isoprene can be biologically produced. To solve this problem as described herein, an isoprene synthase with improved solubility may be expressed in a host (e.g. a bacterial host).
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